The aim of the proposed study is to derive lines of T lymphocyte precursors and to establish their use in studying mechanisms of thymic development. This methodology will enable us to gain insight into fundamental processes that create and sustain a functional immune system. The knowledge of the mechanisms involved is crucial not only to our understanding of human and animal health and illness, but will also help to develop novel approaches to fight auto-immune diseases. A large body of work has been devoted to the study of thymic education. However, several aspects of T lymphocyte differentiation still remain unexplained. Studies aimed at addressing these issues have been hampered by the inaccessibility of significant numbers of undifferentiated T cell precursors. In this proposal, we describe experiments utilizing in vitro model systems that will allow the use populations derived from in vitro differentiated embryonic stem(ES) cells as source of T lymphocyte precursors. The advantage of using ES-derived cells are several fold. First, we can generate large numbers of these cells reproducibly. Second, we can introduce genetic changes in the ES cells and analyze their effects within the differentiating progeny. The developmental potential of these populations will be fully characterized and their useful in looking at thymic differentiation will be determined. Since we have also identified and characterized cell lines that can induce clonal deletion, we will be able to look at interactions between TCR and MHC of different affinity which ultimately are responsible for inducing thymic tolerance.